Recent intense interest in the use of rapid genetic analysis as a tool for understanding biological processes (Wodicka et al., J. Nat. Biotechnol. 15:1359-1367 (1997); Iyer et al., Science 283:83-87 (1999)), in unlocking the underlying molecular causes of disease, and in the development of biosensors, has led to a need for new sensitive and arrayable chip-based analytical tools. Of high importance is the need for techniques that do not require labeling of the target sample (Sando et al., J. Am. Chem. Soc. 124:2096-2097 (2002), since that increases the time, cost, and potential for error inherent in the analysis. In the context of solution-phase assays, the molecular beacon concept has proven itself to be both sensitive and reliable (Broude, Trends Biotech. 20:249-256 (2002); Dubertret et al., Nature Biotech. 19:365-370 (2001)). Molecular beacons consist of a DNA hairpin functionalized at one end with a fluorophore, and at the other with a quenching agent (Tyagi et al., Nat Biotechnol 14:303-308 (1996); Joshi et al., Chem. Commun. 1(6):549-550 (2001)). In the absence of the target DNA sequence, the quencher is brought in close proximity to the fluorophore, and no signal is generated. Addition of the target sequence leads to hairpin unfolding, concomitant duplex formation, and signal generation.
Although a few reports of surface-immobilized molecular beacons have appeared in the literature (Fang et al., J. Am. Chem. Soc. 121:2921-2922 (1999); Wang et al., Nucl. Acids. Res. 30:e61 (2002)), it is believed that these approaches employ an attached single molecule as quencher, while the material on (or in) which the hairpin is immobilized serves only a passive role. As part of a general program aimed at developing “label-free” optical biosensors (Chan et al., J. Am. Chem. Soc. 123:11797-11798 (2001)), it was of interest, therefore, to investigate whether the substrate material itself could be used as a quenching agent.
When attempting to adapt the work of Dubertret et al. (Nature Biotech. 19:365-370 (2001)) by attaching fluorophore-functionalized DNA hairpins to a flat gold surface rather than a gold nanoparticle, as described by Dubertret et al., the inventors of the present application obtained a device that was not functional, presumably because of steric crowding. The gold nanoparticles used by Dubertret et al. contained only a single hairpin per particle; whereas multiple hairpins were bonded to the flat gold surface.
The present invention is directed to overcoming these and other deficiencies in the art.